Bioresource Technology 184 (2015) 363–372
Contents lists available at ScienceDirect
Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech
Review
Extremophilic micro-algae and their potential contribution
in biotechnology
Prachi Varshney a,b,c, Paulina Mikulic b, Avigad Vonshak d, John Beardall b, Pramod P. Wangikar a,⇑
a
Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India
School of Biological Sciences, Monash University, Clayton, Victoria 3800, Australia
c
IIT Bombay Monash Research Academy, CSE Building, 2nd Floor, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India
d
Jacob Blaustein Institutes for Desert Research, Ben Gurion University, Sede Boqer Campus, 84990, Israel
b
h i g h l i g h t s
Extremophilic micro-algae have a potential role in the biotechnology industry.
We consider extremes of temperature, light, CO2, pH, salt and metal in this review.
We present phylogenetic analysis of extremophilic microalgae.
We discuss organisms’ adaptive mechanisms to tackle these stresses.
Physiology, metabolic engineering and molecular biology need further studies.
a r t i c l e
i n f o
Article history:
Received 6 September 2014
Received in revised form 5 November 2014
Accepted 7 November 2014
Available online 15 November 2014
Keywords:
Green algae
Thermophile
Acidophile
Psychrophile
Halophile
a b s t r a c t
Micro-algae have potential as sustainable sources of energy and products and alternative mode of agriculture. However, their mass cultivation is challenging due to low survival under harsh outdoor conditions and competition from other, undesired, species. Extremophilic micro-algae have a role to play by
virtue of their ability to grow under acidic or alkaline pH, high temperature, light, CO2 level and metal
concentration. In this review, we provide several examples of potential biotechnological applications
of extremophilic micro-algae and the ranges of tolerated extremes. We also discuss the adaptive mechanisms of tolerance to these extremes. Analysis of phylogenetic relationship of the reported extremophiles suggests certain groups of the Kingdom Protista to be more tolerant to extremophilic conditions
than other taxa. While extremophilic microalgae are beginning to be explored, much needs to be done
in terms of the physiology, molecular biology, metabolic engineering and outdoor cultivation trials before
their true potential is realized.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Atmospheric carbon dioxide levels have been rising rapidly for
the past 200 or so years (Falkowski et al., 2000). This post industrialization effect has been attributed primarily to anthropogenic CO2
emissions caused by combustion of fossil fuels to meet our energy
demands. These include direct energy consumption (such as electricity and transport) as well as indirect consumption for production and processing of various materials (such as steel, cement
and plastic) used by mankind. Clearly, alternate sources of energy
and products are needed that are carbon neutral and sustainable.
Furthermore, with predictions that the world population will have
⇑ Corresponding author. Tel.: +91 2225767232.
E-mail address: [email protected] (P.P. Wangikar).
http://dx.doi.org/10.1016/j.biortech.2014.11.040
0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
increased by another 2 billion by 2050, a major challenge for the
planet is to provide enough food for its population. Current estimates indicate that sufficient water and arable land is not available
to support such a demand (Fedoroff et al., 2010). Photosynthetic
organisms such as cyanobacteria and eukaryotic algae have the
potential to meet a significant fraction of the requirements of
energy, products, food and animal feed. For ease of reference, while
recognizing their phylogenetic diversity, we loosely refer to these
organisms collectively as micro-algae in this review. Micro-algae
grow much faster and show greater photosynthetic efficiency compared with land plants. Average areal biomass productivities of up
to 20 kg/m2/year have been reported for micro-algal mass cultures,
with a potential for further improvement with strain selection,
strain improvement and process engineering (Williams and
Laurens, 2010). Moreover, micro-algae have the potential to
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P. Varshney et al. / Bioresource Technology 184 (2015) 363–372
produce materials that are of commercial interest, such as astaxanthin (Fan et al., 1998) and long chain omega-3 polyunsaturated
fatty acids (Khozin-Goldberg et al., 2011). Of equal importance is
the possibility that micro-algal biomass offers an additional mode
of agriculture that will provide food and animal feed produced on
marginal land and using marginal water resources so as not to
compete with resources utilized in conventional agriculture.
A key challenge in mass culturing of micro-algae is to find
strains that not only produce marketable products or biomass for
energy and alternative agriculture, but also grow well under industrially relevant outdoor conditions. These may include the need for
growth under (i) high (or low) temperatures due to local climatic
conditions or bubbling of hot flue gases, (ii) wide temperature variations over a diurnal cycle typically seen in desert conditions, (iii)
high light and UV radiation when using solar radiation to drive
photoautotrophic growth, (iv) high CO2 while bubbling flue gases
or limiting CO2 while growing with ambient CO2 if no source of
CO2 supplementation is available, (iv) local water conditions such
as high salt content (e.g., seawater), alkaline or acidic pH and metal
and organic carbon content originating either from local water
bodies or from industrial wastewater that needs to be used for
algal growth. Some of these can be considered extreme conditions,
as most organisms will not survive in such environments. In view
of this, extremophilic micro-algae have the potential to play an
important role in the eventual commercial exploitation of microalgae based biofuels, bioproducts and agriculture.
1.1. Definition of extremophiles
Most organisms have evolved under relatively benign climates
and are not normally able to survive in extremes of environment
such as temperature, pH or in the presence of xenobiotics. However, there are areas on Earth where environmental conditions
are beyond the normal limits for growth and can thus be considered as extreme. Thus organisms that can cope with extremes of
pH, temperature, pressure, and salinity have all been considered
extremophiles. Sometimes these organisms possess additional
qualities such as the ability to cope with very high levels of gases
such as CO2, or grow in the presence of high concentrations of metals and some can thrive in combinations of more than one stress
(polyextremophiles). Some organisms even possess a remarkable
ability to grow under very high ionizing radiation levels and to
accumulate radionuclides (Rivasseau et al., 2013). It is our contention that some of these properties may be of use in biotechnological applications. While prokaryote extremophiles have been of
undoubted value to biotechnology to date, we here concentrate
on the potential application of phototrophic micro-algae in
biotechnology.
Spirulina, a filamentous cyanobacterium that blooms in alkaline
lakes with high pH in the range of 9–11 (Silli et al., 2012). Dunaliella is used as a natural source of b-carotene while Spirulina has a
market as a food and feed additive in human and animal nutrition.
A key factor in the commercial success of these two species is their
ability to grow under specific extreme conditions that help in
reducing the contamination by other algal species (Avron and
Ben-Amotz, 1992). Under mass culturing conditions, these species
are reported to grow at 10–60 g m2 day1. In view of this, extremophilic micro-algae may offer the following advantages in biotechnological applications.
2.1. Ability to grow under local climatic conditions and exclude
potential contaminants
This involves the selection of micro-algae for its ability to grow
under extreme conditions. This will not only optimize biomass
production but also minimize contamination by other algal species. These extreme conditions may include high daytime temperatures, bubbling with flue gases or using water of specific quality
(e.g., high salinity). The extreme conditions will need to be in sync
with the local climatic conditions and water quality to minimize
costs involved in maintaining such conditions. For example maintaining high salinity will require a high cost in medium preparation
unless seawater is used. Another challenge will be the need to
maintain the level of salinity within acceptable bounds and avoid
increases due to evaporation (or dilution by rainwater).
2.2. High value products from extremophilic micro-algae
Extremophiles have developed special mechanisms that allow
the cell to grow and thrive under extreme conditions. The most
common example is accumulation of glycerol as an osmo-regulant
in Dunaliella or the accumulation of b-carotene as a protective
agent against excess light. Such a phenomenon is the basis for
the development of the mass culturing of Haematococcus pluvialis
for the extraction of astaxanthin (Fan et al., 1998). Similar cases
can be found in micro algae that grow in snow and as a result have
had to develop a unique membrane structure to maintain their fluidity or protect the cell from freezing damage, or in the case of algal
species that thrive in hot springs and represent a potential source
of enzymes that are resistant to high temperatures. This approach
requires the development of an intensive collecting and screening
protocol that will identify the potential strains and then go into the
process of developing the biotechnology for mass culturing of the
selected strain. Representative examples of potential biotechnological applications of micro-algae from extreme temperatures are
presented in Table 1.
2.3. Sources of genes that yield products of interest
2. Role of extremophilic micro-algae in biotechnology
Despite their commercial potential having been recognized for
over 50 years, the number of micro-algal species that are currently
produced on a large scale in a sustainable economic process is limited (Richmond, 2004). A major constraint in creating a new flourishing, micro-algae based, agro biotechnology lies in achieving
large-scale under outdoor conditions (Torzillo et al., 2003). High
light, temperature, seasonal and diurnal fluctuations in light and
temperature and contamination by other organisms affect growth
and productivity in outdoor algal ponds (Vonshak and Richmond,
1988). Of the few micro-algal strains that have reached a stage of
being a commercially traded product, two are extremophiles. The
first example is of Dunaliella, a green unicellular micro-algae isolated from high salinity water bodies with NaCl concentrations
exceeding 3 M (Borowitzka and Huisman, 1993). The other one is
The third approach, and one that is gaining more interest in
recent years, is to view the extremophiles more as a source of
genes that can be isolated and cloned into other organisms that
are more easily mass cultured and in some cases even have the
capacity to be grown heterotrophically.
The real challenge is to identify a product that will represent a
unique advantage to be produced from the microbial biomass and
represent a true economic advantage over the traditional sources
of conventional agriculture, chemical synthesis or standard fermentation technology. This paper gives an overview of extremophilic micro-algae (cyanobacteria and eukaryotic microalgae) and
asks the question as to whether adaptations to extreme environments in these organisms confers a particular ability to cope with
the constraints of growing in mass cultures or induces production
of unique compounds of potential use in biotechnology.
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Table 1
Potential biotechnological applications of some psychrophilic and thermophilic algae and cyanobacteria. Representative applications are listed based on specific reports.
1
Group
Product/application
Psychrophilic algae (snow algae)
Chlamydomonas nivalis
Raphidonema sp.
Mesotaenium berggrenii
Chloromonas sp. (CCCryo 020–99)
Nostoc commune
Green algae
Green algae
Green algae
Green algae
Cyanobacteria
Astaxanthin
a-Tocopherol (vitamin E) and xanthophyll cycle pigments
Sucrose, Glucose, Glycerol
Glycerol
Myxoxanthophylls and canthaxanthin
Thermophilic cyanobacteria and algae
Phormidium sp.
Thermosynechococcus elongatus BP-1
Desmodesmus sp. F51
Galdieria sulphuraria 074G
Galdieria sulphuraria CCMEE 5587.1
Chlorella sorokiniana UTEX 2805
Desmodesmus sp. F2 and F18
Cyanobacteria
Cyanobacteria
Green algae
Red algae
Red algae
Green algae
Green algae
Thermostable restriction enzyme
Thermostable phosphate kinase
Lutein (xanthophyll) [feed additive & natural colorant for pharmaceuticals]
Blue pigment phycocyanin (PC) used as a fluorescent marker in histochemistry
Wastewater treatment
Wastewater treatment (Ammonium removal)
High lipid content (up to 58% of dry biomass)
References1
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
The references cited here have been listed in Supplementary information.
3. Constraints in algal biotechnology – how can extremophiles
fill the gap?
For biotechnological purposes, micro-algae are generally grown
either in closed photobioreactors or in open raceways. Although
phototrophic cultures are frequently light limited at depth, at the
surface of cultures they can be exposed to the full force of the
sun. In open raceways where there is no screening, this can also
involve exposure to UV radiation. In closed systems, oxygen
build-up in cultures can, in combination with other stressors lead
to significant formation of damaging reactive oxygen species
(ROS). Freshwater is often in scarce supply, especially in areas
where solar radiation is high enough to minimize light limitation
of algal mass cultures. Access to organisms that have a high tolerance to brines or hypersaline environments can get around constraints on freshwater supply. In dense cultures, inorganic carbon
can be rapidly depleted, thus limiting photosynthesis and growth.
This is overcome in most algae and cyanobacteria by operation of a
CO2 concentrating mechanism. In practice, CO2 levels in raceways
and PBRs can be maintained by sparging with CO2 – this being
especially important in the use of photoautotrophs to remediate
CO2 emissions. Given that CO2 in waste gases from power-plants
and other industrial applications can be very high (>12% for
power-plant flue effluent) an ability to tolerate high CO2 and the
low pH values that result can be important.
4. Phylogeny of extremophilic micro-algae
Micro-algae belong to a number of evolutionarily distinct major
groups within the Kingdom Protista (see e.g. Yoon et al., 2004).
Cyanobacteria are an ancestral clade of prokaryotes originating
2700–3500 million years ago (MYA) (Falkowski et al., 2004) and
are also the progenitors of plastids in photosynthetic eukaryotes.
For the purpose of this review, it is apparent that in oxygenic
phototrophs, extremophiles can be found among cyanobacteria,
red algae (especially in the Cyanidiales) and green algae (chlorophytes) (Fig. 1). More recent, but ecologically important, taxa such
as the diatoms and prymnesiophytes (members of the Heterokont
algae) and the dinoflagellates do not have any reported extremophilic representatives with the exceptions of the diatom Pinnularia
sp., found in some low pH freshwaters (Aguilera et al., 2006) and a
reasonably extensive flora of psychrophilic species such as the
polar/sea-ice diatoms Entomoneis and Fragilariopsis (Seckbach,
2007). Among the photosynthetic eukaryotes, the red algal Cyanidiales are an ancient lineage with the majority of high temperature and acid-tolerant eukaryotic alga belonging to this branch.
The chlorophytes are not reported to tolerate temperatures above
55 °C while red algae, especially the Cyanidiales, tolerate up to
60 °C, though, as we will see, green algae exhibit tolerances to
other extremes. Some cyanobacteria are reported to tolerate up
to 74 °C. Higher plants are believed to have evolved 470 MYA
and are not known to tolerate temperatures above 50 °C, though
some species have other extremophile characteristics such as desiccation tolerance or capacity for metal hyperaccumulation, that
are outside the scope of this review.
5. Low and high temperature tolerant algae
Temperature is an important growth-determining factor for
organisms. Extreme temperatures such as the extreme cold of
the frozen deserts of Antarctica and temperatures above boiling
in the hot springs of Yellowstone National Park present challenging
growth environments to biota. Consequently, species diversity is
quite low in these harsh environments. Yet there are organisms
which thrive and complete their life cycle at such extreme
temperatures.
Depending upon the optimal growth temperature, species can
be broadly classified as (1) Psychrophiles growing optimally below
a temperature of 15 °C, (2) Thermophiles growing at temperatures
>50 °C and (3) Mesophiles growing best at intermediate temperature. A fourth class, Hyperthermophiles, have optimum temperatures of >80 °C. As observed above, most of the psychrophilic and
hyperthermophilic organisms belong to archaeal or bacterial
domains (D’Amico et al., 2006), Fig. 2 places some representative
micro-algae on a temperature scale based on their optimal temperatures for growth. Although there are photosynthetic prokaryotes
such as a few cyanobacteria and some purple and green sulfur bacteria which grow at temperatures up to 74–75 °C, the upper temperature limit for eukaryotic algae is 62 °C (Rothschild and
Mancinelli, 2001) (Fig. 2). No photosynthetic organisms have been
reported that grow beyond 75 °C, possibly due to the instability of
chlorophylls beyond this threshold. At low extremes, some snow
and ice algae grow even at 1 °C (Fujii et al., 2010).
5.1. Psychrophiles
Extremely cold regions of the Arctic and Antarctic, and moderately cold mountainous regions are dominated by psychrophiles
and psychrotrophs. Psychrophiles grow in permanently cold environments whereas psychrotrophs are not completely adapted to
cold and sometimes have upper temperature limits of >20 °C. The
dominant phototrophic cyanobacteria in the Antarctic region
include the genera Oscillatoria, Phormidium, and Nostoc (D’Amico
et al., 2006) (Fig. 2), though there are a great many cold tolerant
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Fig. 1. Phylogeny of photosynthetic extremophiles. Extremophiles are found predominantly in the red algal (Cyanidiales) and chlorophyte lines of eukaryotes and the
ancestral cyanobacterial line. A few angiosperms (Ang.) have extreme desiccation tolerance, as do some bryophytes. (Gymno = gymosperms; Heterokonts include diatoms,
chrysophytes and phaeophytes; haptophytes include the ecologically important coccolithophores). Not all algal lines are shown (dinoflagellates in the Alveolates are missing,
but have no extremophile representatives) and timing of events is approximate. Redrawn after (Yoon et al., 2004).
Fig. 2. Temperature limits for microalgal life. Optimal reported growth temperature for representative microalgae are shown. Red algae are indicated in red, green algae in
green, blue-green algae (cyanobacteria) in blue, and diatoms in brown. The numbers in parenthesis indicate the reference number as listed in the Supplementary information.
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
diatoms and other psychrophilic eukaryotic algae that are the
major primary producers in polar marine environments. Snow
algae, which grow in cold regions, create huge blooms of
macroscopically visible pigmentation on the snow with different
colors such as red, orange, pink and green. The green color is a
result of actively dividing sexual and asexual stages of cell,
P. Varshney et al. / Bioresource Technology 184 (2015) 363–372
whereas the red color is due to carotenoids, produced especially in
resting stages. Common eukaryotic snow algae genera are Chlamydomonas, Chloromonas, Microglena, Chlorella and Scenedesmus.
Psychrophiles have the ability to overcome adverse effects of
low temperatures that cause an exponential drop in biochemical
reaction rates. Thus, enzymes of these microorganisms are adapted
to cold temperatures and have high catalytic efficiency at low temperature relative to their mesophilic and thermophilic counterparts. They are also capable of tolerating the increased water
viscosity, which roughly doubles in going from 37 °C to 0 °C. Psychrophiles can potentially be used for the production of detergents,
fine chemicals, in food industry and for the treatment of hydrocarbon contaminated soil in Antarctica (Hoover and Pikuta, 2010).
Psychrophiles maintain their membrane fluidity at low temperature by incorporating a higher level of polyunsaturated fatty acids
(PUFAs) in membrane lipids, thereby making them a potentially
useful source of PUFAs for nutraceutical products such as eicosapentaenoic acid, arachidonic acid and docosahexaenoic acid.
5.2. Thermophiles
Though thermophilic life has been known for many years,
reported studies were very limited until the thermophilic bacterium Thermus aquaticus was discovered in 1969 in the Mushroom
Spring of Yellowstone National Park (Brock and Freeze, 1969).
Since then, hundreds of thermophilic species have been identified
in all the three domains of life, namely bacteria, archaea and eukarya. Thermophiles have thermostable enzymes with temperature
optima for activity as high as 90 °C. Organisms that have adapted
to extremes of temperature have been the subject of some interest
for biotechnology (and it must be pointed out that thermo- and
psychro-philes are often subject to additional stresses such as very
high irradiance, and thus qualify as polyextremophiles). For
instance, Leya et al., 2009 have indicated the psychrophile Raphidonema may be a good source of a-tocopherol and various carotenoids, while the snow algae Chloromonas nivalis and
Chlamydomonas nivalis synthesize high levels of astaxanthin
(Remias et al., 2010, 2005). Further examples are given in Table 1.
Biotechnological interest in thermophilic microalgae is more centered on their use as sources of thermostable enzymes. While thermostable restriction enzymes are usually sourced from the
bacterium T. aquaticus, the cyanobacterium Phormidium has also
been shown to produce a similar enzyme. Thermophilic alga Galdieria sulphuraria (a red alga) and Desmodesmus (a green alga) have
both been investigated for useful pigment production. G. sulphuraria has also been tested for its ability to remove nutrients from primary wastewater effluent. (Table 1).
6. Ability to grow under low and high CO2 levels
In dense mass cultures, intense photosynthetic activity
decreases the dissolved inorganic carbon (DIC) concentration significantly. In poorly buffered systems, CO2 uptake by the culture
causes the pH to rise, and values of pH 9–10 are not uncommon.
This rise in pH in turn leads to a decrease in the CO2 to bicarbonate
ratio as well as the decrease in absolute CO2 concentration. Thus,
DIC levels in cultures, even if quite well aerated, are frequently
below atmospheric-equilibrium. For instance, Williams and
Colman, 1996 showed that the DIC concentration in cultures of
the acidophile Chlorella saccharophila dropped from 450 lM (air
equilibrium) to below 30 lM over 3 days under low aeration rates.
This decrease in DIC below air-equilibrium can impose limitations
on diffusive supply of CO2.
While it is possible in some instances to rectify DIC depletion by
using sources of high CO2, such as flue gases, mass algal cultures in
367
remote areas without a CO2 source nearby will be severely carbon
limited. In these circumstances, efficient use of the available inorganic carbon by the algal/cyanobacterial cells would be
advantageous.
6.1. RuBisCO and CO2 concentrating mechanisms (CCMs) under
extreme conditions
All cyanobacteria and eukaryotic microalgae (and indeed
almost all autotrophs) rely on the enzyme ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) and the Photosynthetic
Carbon Reduction Cycle (PCRC; the Calvin–Benson–Bassham Cycle)
for net assimilation of inorganic carbon to organic matter. In
addition to the fixation of CO2 to the acceptor molecule ribulose1,5-bisphosphate (RuBP), giving rise to 2 molecules of 3-phosphoglycerate, RuBisCO also catalyses an oxygenase reaction, the
products here being one molecule each of phosphoglycerate and
phosphoglycolate. One of the 2 carbons in phosphoglycolate can
be recovered and converted to phosphoglycerate by the reactions
of the photosynthetic carbon oxidation cycle (PCOC) in the process
of photorespiration, but one carbon is lost as CO2 and represents a
potentially significant inefficiency in the carbon assimilatory process (Giordano et al., 2005). The carboxylase and oxygenase activities of RuBisCO are competitive and dependent on the ratio of
oxygen and CO2 at the enzyme active site, according to Eq. (1)
Srel ¼
kcat ðCO2 Þ
K 1=2 ðCO2 Þ
kcat ðO2 Þ
K 1=2 ðO2 Þ
ð1Þ
where the selectivity factor Srel defines the ratio of rates of carboxylase to oxygenase reactions and the kcat and K½ values correspond
to the maximum specific reaction rates and half-saturation concentrations for the respective substrates. Different forms of RuBisCO
that have evolved in autotrophic organisms. Most microalgae and
cyanobacteria have forms of RuBisCO with 8 large and 8 small
sub-units (L8S8) and are known as Form I (marine Synechococcus
and all Prochlorococcus species possess Form IA RuBisCO while most
freshwater cyanobacteria and some eukaryotic algae have Form IB
and red algae, cryptophytes, haptophytes and diatoms all have
Form ID). Form II RuBisCO comprises only 2 large subunits (L2)
and is found in dinoflagellates and Chromera veria. Form III is found
only in archaea and will not be discussed further. The evolutionary
origins of the different forms of RuBisCO are discussed in detail by
(Raven et al., 2012).
These forms of RuBisCO possess different kinetic properties in
relation to affinity for CO2 (K½(CO2)) and specificity for CO2 vs O2
(Srel, Eq. (1)) (Raven et al., 2012, 2011; Whitney et al., 2011). Values
for these parameters are highly variable (Giordano et al., 2005):
Miller et al. (2007) report the highest recorded value for
K0.5(CO2) for a Form 1 RuBisCO of 750 lM for the marine cyanobacterium Prochlorococcus marinus whereas green algae have RuBisCOs with K½(CO2) 30 lM. Form II RuBisCOs have very low Srel
values and dinoflagellates might struggle to perform net C assimilation under air equilibrium CO2 levels (Giordano et al., 2005). The
general trend across all autotrophs is that a low K½(CO2) and high
Srel are correlated with a low kcat(CO2), and vice versa (Raven et al.,
2012).
RuBisCO evolved at a geological time in which CO2 levels were
very much higher than at the present day. The subsequent longterm drop in CO2 levels and rise in oxygen has led to a situation
where competition between O2 and CO2 has become restrictive
to net carbon fixation. Thus, in general (and there are exceptions
– see below), at present-day dissolved CO2 levels of 15 lmol L1
(the exact concentration depending on salinity and temperature),
organisms will have RuBisCOs operating well below maximum
capacity if the internal CO2 is in equilibrium with (or lower than)
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the external CO2. However, cyanobacteria and algae have mechanisms to enhance CO2 levels at the active site of RuBisCO (CCMs;
see recent reviews by (Giordano et al., 2005; Raven et al., 2012).
There is evidence that there is an inverse relationship between
kinetic characteristics of RuBisCOs and the extent to which algae
and cyanobacteria express CCMs, such that species with high Srel
RuBisCOs have lower levels of CCM expression than do species
with low Srel RuBisCO (Tortell, 2000). Thus there seem to be two
approaches to the ‘‘low CO2’’ problem, one involving evolution of
active CCMs and the other involving the evolution of RuBisCOs
with higher affinity for CO2. CCM activity is strongly regulated by
environmental factors (Raven et al., 2011). Foremost among these
is the CO2 level in solution (though cyanobacterial CCMs seem to
be controlled directly by HCO
3 concentrations instead (Mayo
et al., 1986).
Thus high salinities, in which dissolved CO2 at air equilibrium is
lower, resulted in increased CCM activity in the halophile Dunaliella salina (Booth and Beardall, 1991). Low temperatures result in
increased solubility of CO2 and higher inorganic C concentration,
and also influence the ratio of carboxylase to oxygenase activities
in favor of carboxylation (Descolas-Gros and de Billy, 1987), but
studies to date suggest that polar species adapted to very cold
environments do not show diminished CCM activity compared to
their temperate counterparts (Beardall and Roberts, 1999). On
the other hand, some thermophilic (and acidophilic) extremophile
algae have evolved RuBisCOs with very high affinity (low K0.5) for
CO2; Uemura (1997) report K0.5 CO2 values of 6.6 and 6.7 lM for
the red algae Galdieria partita and Cyanidium caldarium, respectively, with specificity factors over 220, compared to a value for
143 for RuBisCO from the temperate red alga Porphyridium purpureum (K0.5 CO2 = 22 lM). Other, non-thermophilic, acidophiles such
as C. saccharophila and Chlamydomonas acidophila have retained
CCMs (Spijkerman, 2005; Beardall 1981); and so has the acidophilic red alga Cyanidioschyzon merolae (Zenvirth et al., 1985),
although there are no data on the kinetics of RuBisCO from these
species. It is apparent from the above discussion that extremophiles may have representatives with especially good affinities
for inorganic carbon which offer a high capacity for overcoming
potential limitations of growth at high cell density in mass
cultures.
6.2. Tolerance to high CO2 levels
In order to (a) prevent CO2 limitation and (b) to carry out remediation of CO2 emissions from industry, there is considerable interest in using industrial flue gases to supplement CO2 supplies to
microalgal cultures. Since flue gases contain CO2 at high levels
(typically 10–12% compared to 0.04% in the present day atmosphere), using micro-algae tolerant to such concentrations (and
the decrease in pH they engender) is of considerable importance.
Flue gases also contain SO2 which, despite efficient scrubbing technologies, may still reach 2400 ppm in the CO2 stream (Lee et al.,
2009). Species tolerant to high CO2 and SO2, together with low
pH could thus be highly desirable. Many green algae and some cyanobacteria are tolerant to quite high CO2 levels (>20%) and in the
case of Chlorella T-1 and Scenedesmus strain K34 (see Table 2) will
survive exposure to 100% CO2 in a gas stream. However, few of
these are also tolerant to the high temperatures such gases can
potentially attain when they leave the flue – the cyanobacterium
Chroococcidiopsis and the green alga Chlorella T1 are possible
exceptions. On the other hand species such as C. caldarium, originating as it does from hot springs where high levels of subterranean gases bubble up from underground, have exceptional
thermal and CO2 tolerance. This suggests that species from hot
springs and volcanic seeps may be good candidates for growth
on flue gases.
Table 2
Representative examples of microalgae that can tolerate high CO2 levels. Some strains
also tolerate high temperatures. Note that the optimum growth temperatures and
CO2 levels may be different than those listed as the tolerated values.
Algal species
Red algae
Cyanidium
caldarium
Green algae
Chlorella sp. T-1
Scenedesmus sp.
strain K34
Chlorella sp. K35
Chlorella ZY-1
Chlorella sp. UK001
Chlorella vulgaris
UTEX 259
Chlorella kessleri
Scenedesmus
obliquus
Nannochloris sp.
(NOA-113)
Monoraphidium
minutum
(NREL strain
MONOR02)
Cyanobacteria
Chroococcidiopsis
strain TS 821
Synechococcus
elongates
Chlorogleopsis sp.
(SC2)
1
Temperature
tolerance (°C)
Maximum CO2
tolerance (v/v) (%)
References1
56
100
(13)
35–45
40
100
100
(14)
(15)
45
40
30 (up to 45)
20
80
70
40
30
(15)
(16)
(17)
(18)
50
50
18
18
(19)
(19)
25
15
(20)
25
13.6
(21)
50
80
(22)
60
60
(23)
50
5
(24)
The references cited here have been listed in Supplementary information.
7. Tolerance to high levels of solar radiation
One of the major constraints in algal and cyanobacterial mass
culture is the limited penetration of photosynthetically active radiation (PAR) through the cultures. Approximately 90% of the incident photons are absorbed by the uppermost 10% of the culture.
The remaining culture volume (90%) is thus using photons very
efficiently, but is severely light limited (Ritchie and Larkum,
2012). Changes in light availability over the seasons and with latitude may restrict sustainable, year-long, high yields to lower latitudes (<35°, Williams and Laurens, 2010). At these low latitudes,
solar radiation is much higher than at high latitudes and can
potentially cause severe photoinhibition in cells in the upper
regions of cultures.
Similarly, UVB fluxes are considerably higher in the tropics than
at high latitudes, though UVBR fluxes at high latitudes are more
seasonally variable (Whitehead et al., 2000). UV radiation has a
range of inhibitory effects on the physiological activities of algae
and cyanobacteria (Beardall et al., 2009). These include inhibition
of damage to DNA and to light transduction and carbon assimilation mechanisms as well as inhibition of nutrient uptake. Algae
have a range of physiological strategies to cope with excess PAR
and UVB. These include a range of antioxidants, mechanisms for
scavenging reactive oxygen species (ROS), non-photochemical
quenching for excess light dissipation, and mechanisms for repairing damage from excess PAR and UVB, though these can be energetically costly (Raven and Ralph, 2014). Algae and cyanobacteria
also have a range of inducible UV-screening compounds, which
can reduce UV-A and UV-B absorbance and minimize damage
(Rozema et al., 2002). Using cyanobacteria as sources for production of UV-sunscreens has been proposed by Browne et al. (2014).
Given that algae and cyanobacteria have evolved mechanisms
to cope with high levels of PAR and UV radiation, it is likely that
P. Varshney et al. / Bioresource Technology 184 (2015) 363–372
organisms from environments with very high solar radiation (low
latitude desert regions, with little screening from clouds) may have
evolved exceptional capacities to cope with such stresses. There
are certainly reports of positive effects of UV-B on recovery of algae
from photoinhibition and stimulation of photoprotection mechanisms (Flores-Moya et al., 1999). Thus organisms that are habitually exposed to high UVB might be expected to show especially
high capacity to withstand this stress. Not only would such algae
and cyanobacteria be capable of growing well under extremes of
PAR and UVB, they might also be excellent sources of antioxidants
and protective compounds. Examples of the latter include b-carotene production by the green alga Dunaliella tertiolecta and astaxanthin formation by Haematococcus, as well as production of the
antioxidant a-tocopherol by Raphidonema, as mentioned above.
Formation of reactive oxygen species (ROS) is a common
response to a range of stresses and can be a by-product of both respiration and photosynthesis. In photosynthetic metabolism ROS
arise when there are insufficient electron sinks available so electrons from the light harvesting process are passed on to oxygen.
Cells possess a range of antioxidative mechanisms, which ensures
that ROS molecules are reduced before they can cause serious oxidative damage to cell components. When ROS levels exceed the
antioxidative capacity of cells, oxidative damage will result in
damage, causing alteration of cell structure and symptoms of oxidative stress.
Organisms commonly exposed to very high light, especially if
combined with other stresses, are particularly likely to produce
high levels of ROS. However, many extremophilic organisms possess remarkable abilities to withstand these stresses. Of particular
note are those cyanobacteria and algae found in biological sand
crusts (BSCs). For example the cyanobacterium Microcoleus up-regulates light energy quenching mechanisms, allowing it to continue
to photosynthesise at very high light without photoinhibition
(Ohad et al., 2010). Among eukaryotes, a newly described Chlorella
species, C. ohadii, is well adapted to the harsh conditions of the
BSCs and thrives at extremely high light (Treves et al., 2013).
Organisms from environments such as BSCs may thus be ideal
candidates for biotechnological exploitation as well as potentially
acting as a source of genes to improve the performance of nonextremophiles under stress conditions.
369
8. Tolerance to high levels of metals and low pH
Naturally occurring acidic environments harbor a diverse range
of acidophilic microorganisms, including algae, which tolerate, and
in some cases thrive at pH values most microorganisms would not
cope with (Souza-Egipsy et al., 2011). Such naturally occurring
environments are a result of the leaching of substances, for example fumic and fulvic acids from podocarp rainforests, or volcanic
activity (Collier et al., 1990). Highly acidic environments also result
from anthropogenic activity. Highly acidic environments facilitate
metal solubility, resulting in an environment in which metal-tolerant acidophiles reside (Novis and Harding, 2007). Rio Tinto, a river
in southwestern Spain, is an example of a system with low pH and
metal levels toxic to most aquatic organisms, yet it harbors a
diverse range of eukaryotic microorganisms that are the main contributors to the biomass of the river (Souza-Egipsy et al., 2011). An
acido-thermophile Cyanidioschyzon sp. that lives in algal mats in
environments high in arsenic, in conjunction with Cyanidium and
Galdieria (order Cyanidiales), plays a role in biotransforming
arsenic present in their environment (Qin et al., 2009). Fig. 3 provides a glimpse of micro-algal representatives that grow optimally
under acidic or alkaline pH.
Many microorganisms that grow in such environments grow as
biofilms. It is thought that the formation of extracellular polymeric
substances (EPS) is potentially responsible for the detoxification of
the metal as well as an aid for the formation of the biofilm (GarcíaMeza et al., 2005). Other methods of dealing with metal toxicity
are exclusion from the cell, binding to the cell wall, sequestration
within the cells either by incorporation into cellular processes, or
by complexation with organic compounds and storage in the vacuole (Rai and Gaur, 2001).
Photosynthetic organisms are of great interest in toxicological
studies, as they are the point of entry into the food chain
(Sabatini et al., 2009), and have been used in developing toxicological bioassays (Stauber and Davies, 2000). This ability to accumulate metals, and other pollutants can be harnessed in creating
remediation solutions using actively growing photosynthetic biofilms found in acidic environments with high levels of metals.
Systems using immobilized algae are being developed for treatment of wastewater, due to their photosynthetic capabilities
resulting in the production of useful biomass, as well as the
Fig. 3. pH limits for microalgal life. Examples of known pH limits for representative acidophilic, mesophilic and alkalophilic algae and cyanobacteria are shown with the same
color scheme as in Fig. 2. The numbers in parenthesis indicate the reference number as listed in the Supplementary information. (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of this article.)
370
P. Varshney et al. / Bioresource Technology 184 (2015) 363–372
treatment of wastewater (Mallick, 2002). Membrane transport,
ion-exchange columns, flocculation and other treatments can be
expensive and also produce secondary pollution (Fu and Wang,
2011). Algae grow photosynthetically on limited substrates, and
are therefore efficient and environmentally favorable for remediating point pollution, for example arsenic-contaminated drinking
water in Bangladesh. An optimized and scaled-up phycoremediation system coupled with physico-chemical methods might offer
solutions to larger scale remediation projects (Abdel-Raouf et al.,
2012). An extreme form of metal tolerance is found in those organisms that can grow in the presence of radionuclides and which are
therefore extremely resistant to ionizing radiation. For example
Rivasseau et al. (2013) have isolated Coccomyxa actinabiotis from
a pool used to store spent fuel elements and this is reported to
withstand gamma ray doses of radiation up to 20 KGy and to accumulate, and remove from solution, a range of nuclides including
110m
Ag, 65Zn and 137Cs 60Co, and 238U. Earlier work has also shown
cyanobacteria to possess a degree of radiation resistance (Kraus,
1969). The possibility therefore exists to use phycoremediation of
dealing with radiation contamination in water.
8.1. Adaptations to extremes: low pH and metals
Low pH increases the solubility of metals, and in turn, high
metal levels cause toxicity (Novis and Harding, 2007), either
through molecular mimicry, or competition with other nutrients
within the growth media (Pinto et al., 2003). Often, at polluted
sites where many metals are present, extremophiles have either
a genetic or physiological adaptation that allows them to tolerate
many metals at once, further increasing their applicability to remediation solutions (Rai and Gaur, 2001). Cellular response to a metal
ion can be either exclusion of the metal from the cell, the uptake
and modification of the metal to a less toxic form, followed by
the metal’s transportation out of the cell, or by internal sequestration (Hall, 2002). These processes are a result of a coordinated network of biochemical processes, which increase the cell’s ability to
maintain homeostasis, and minimize oxidative insult as a result of
the production of ROS.
Metal intoxication increases ROS levels as they play a significant
role in many ROS-producing mechanisms, including the Haber–
Weiss cycle, Fenton’s reactions, disruption of the photosynthetic
electron chain leading to O
2 , and reduction of the glutathione pool
(Pinto et al., 2003). ROS also act as signalling molecules that induce
the production of a network of antioxidants, antioxidant enzymes,
and other stress related molecules (Panchuk et al., 2002). Components of the antioxidative response activated by metal stress
include antioxidant enzymes and low molecular weight antioxidants, metal chelating molecules such as phytochelatins, metallothioneins, urate and glutathione.
A well-known limitation of biological-based remediation is the
reliance on natural systems, which are not yet fully understood.
Further elucidating oxidative and stress responses of algae would
aid in optimizing productivity of algal bioprocess systems for metal
remediation. Acidophilic extremophiles are a great tool for such
studies, as they are known for their metal tolerance under already
extreme environments (Whitton, 1970). They have the potential to
provide much knowledge of cellular tolerance mechanisms to
advance and optimize the bioremediation potential of extremophiles, as well as providing many potential species for the construction of bioremediation systems.
ROS signalling pathways and the antioxidative response may
hold the key to understanding how extremophilic organisms tolerate metal oxidative stress. For example, in the extremophilic bacterium Pseudomonas fluorescens, it was shown that a ROS triggered
cascade of biochemical reactions lead to an increased level of
NADPH, therefore an increased reductive capacity and tolerance
Table 3
Examples of micro-algae that are tolerant to high salt levels and some of their
potential products.
1
Species
Product/Application
Reference1
Green algae
Dunaliella salina
Dunaliella bardawil
Dunaliella sp.
Nannochloropsis salina
b carotene (antioxidant)
b carotene (antioxidant)
Glycerol
Higher level of lipids (up to 28%)
(25, 26)
(25)
(27)
(28)
Cyanobacteria
Aphanocapsa
halophytica MN 11
Anabaena sp. ATCC 33047
Cyanothece sp. ATCC 51142
EPS (Exopolysaccharides)/RPS
(Released Polysaccharide)
EPS
EPS
(29)
(30)
(31)
The references cited here have been listed in Supplementary information.
to the oxidative stress inducing Al (Singh et al., 2005). A comparative study of the neutrophilic alga Chlamydomonas reinhardtii and
the acidophilic C. acidophila show the C. acidophila had an increased
basal level of HSP, which is thought to be an adaptive mechanism
to extreme acidic environment (Gerloff-Elias et al., 2006). Another
comparative study of C. reinhardtii and C. acidophila showed that in
face of metal stress, the acidophile produced higher levels of the
antioxidative enzymes ascorbate peroxidase (APX) (Garbayo
et al., 2007). Panchuk et al. (2002) found that in Arabidospsis, the
production of heat shock transcription factors was closely tied with
the production of not only HSP, but also APX. The acidophiles Coccomyxa onubensis and C. acidophila increased their production of
the antioxidants lutein and b-carotene in the presence of copper
at 0.2 mM and 4 mM, respectively (Garbayo et al., 2008; Vaquero
et al., 2012).
There is still much to learn about genes and metabolic pathways involved in metal tolerance and sequestration (Hall, 2002).
Understanding vacuolar transporters functioning in metal sequestration, membrane transporters that aid in phytochelatin and gluthathione transport, and gene-expression induced by metal stress
would greatly aid in identifying hyperaccumulators, and also aid
the engineering of cells with enhanced metal tolerance. Further,
multiple tolerance mechanisms can be introduced to cells to produce highly tolerant photosynthetic organisms to one or more abiotic stressors (Dobrota, 2006).
9. Tolerance to extreme salinity
As has already been flagged, growth under conditions that are
unfavorable to grazers or competing microalgae can be a desired
characteristic for large scale, particularly outdoor, cultures. A number of species of microalgae capable of growth under hypersaline
conditions are well established in biotechnology. Thus the green
alga D. salina (and Dunaliella bardawil, sometimes considered a
strain of D. salina) grow well on very high salinity up to 3 M and
large, open ponds of these algae are used for b-carotene production. Nannochloropsis salina has been grown for lipid production.
A number of cyanobacteria are also capable of growth under high
salinities and Aphanocapsa halophytica and strains of Anabaena
and Cyanothece produce high levels of extracellular polysaccharides under those conditions (Table 3). While the microalgal flora
of hypersaline environments is somewhat restricted, further investigations may uncover additional halophiles or new and desirable
properties of known halophilic species.
10. Concluding remarks and future directions
Extremophilic microalgae have evolved mechanisms that allow
them to tolerate conditions that would be toxic to other organisms.
P. Varshney et al. / Bioresource Technology 184 (2015) 363–372
Bioprospecting for new species/strains may identify yet more useful characteristics for biotechnology. We argue that extremophiles
are currently an under-exploited resource and that further investigations on the following may be needed:
(1) Tolerance mechanisms to environmental stresses.
(2) Suitability of known extremophiles under outdoor
conditions.
(3) Metabolic engineering (Alagesan et al., 2013) of extremophiles; molecular biology tools.
(4) Circadian rhythms in extremophiles, a concept that is widely
researched in mesophilic cyanobacteria (Krishnakumar
et al., 2013).
Acknowledgements
J.B. and P.P.W. gratefully acknowledge the J.S.W. foundation for
financial support. P.V. is grateful to J.S.W. Foundation for providing
a PhD fellowship. A.V. and J.B. acknowledge the support of the Pratt
Foundation.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2014.11.
040.
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